Interferons are a family of inducible, antiviral proteins that are secreted by most animal cells in response to a wide variety of viral and non-viral agents. Interferons have a direct role in antiviral defense in mammals, including humans. They also have been shown to possess immunoregulatory activities and to affect cellular growth. Because of these properties, interferons are currently being tested in the treatment of cancer and immune dysfunctions.
Three distinct major species of interferons have been characterized. Alpha-, or leucocyte interferons are the major species produced by incubating viruses with cells of lymphoid origin. Beta- or fibroblast interferons are the major species produced by nonlymphoid cells upon induction with viral or non-viral agents. Gamma- or immune or type II interferons are the major species produced by lymphocytes (T cells in particular) upon treatment with mitogens, or by sensitized lymphocytes upon treatment with specific antigens.
The three interferon species have very different amino acid sequences and can be distinguished by their physicochemical properties or by specific antisera. For example, antialpha serum does not inhibit the activity of beta- or gamma-interferons.
One of the problems in the clinical application of interferons is that they are in short supply. Purification of native interferons is a complex and expensive procedure. Resolution is often poor and yields are low. Moreover, interferons do not respond well to manipulation (such as that required to purify them) and can be easily inactivated.
Synthetic techniques using either recombinant DNA methods or solid phase peptide synthesis have increased the availability of interferons. However, the recombinant DNA products also need substantial purification (similar to that required for the native products and presenting similar problems) and the peptide-synthesis products are expensive to manufacture because their amino acid chain is quite long (146 amino acids for gamma-interferon).
Therefore, there is a felt need in the art for a peptide having gamma-interferon activity and an amino acid sequence substantially shorter than that of native or recombinant gamma-interferon. Such a peptide could be easily synthesized by solid phase synthesis techniques well-known in the art.
Virtually nothing is known about the existence of biologically active peptide fragments of human gamma-interferon having substantially lower molecular weights. U.S. Pat. No. 4,599,306, issued to Altrock, disclosed a nonadecapeptide whose amino acid sequence duplicated the last 19 amino acid residues of the carboxy terminal of gamma-interferon. However, the peptide was used only to demonstrate and quantitate the binding of a monoclonal antibody to gamma-interferon, and no disclosure was made concerning the biological activities of this peptide.
Rinderknecht et al (J. Biol. Chem. 259:6790-6797, 1984) disclosed that removal of up to 16 amino acids from the carboxyl terminal of gamma-interferon did not affect its biological activity. However, no peptide having less than the remaining 130 amino acids but still retaining biological activities was disclosed.
U.S. Pat. No. 4,604,284, issued to Kung et al, disclosed an active 15 kD fragment of gamma-interferon. Kung et al observed that purified gamma-interferon preparations are not homogeneous, but contained a variety of fragments. This 15 kD gamma-interferon fragment resulted from the removal of C-terminal amino acid residues 132-146 by proteases present in the interferon-containing supernatant. However, no active peptide containing less than 131 amino acid residues was disclosed or suggested.
The present inventor has unexpectedly found a novel peptide having gamma-interferon activity and comprising a sequence of less than about 100 amino acids. More particularly, the present inventor has isolated a novel peptide which has a molecular weight of between about 7,000 and 8,000 Daltons, is immunochemically reactive with gamma-interferon antibodies, and displays antiviral and cytolytic activities. Surprisingly, the novel gamma-interferon peptide of the present invention was not detectable on sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE), i.e., under denaturing conditions. 0n SDS-PAGE, only two gamma-interferon polypeptides were detectable with apparent molecular weights of 52 kD and 28 kD.
The use of the process for purifying the peptide of the present invention, wherein high resolution and speed in the separation of protein molecules was achieved, coupled with the high degree of recovery of biologically-active interferon molecules, allowed for the discovery of this heretofore unrecognized component of the gamma-interferon-containing supernatant.
The present invention has several objects including, but not limited to, the following:
To isolate and characterize a substantially smaller novel polypeptide having gamma-interferon activities;
To purify this peptide without substantially decreasing its activity;
To isolate and characterize a novel polypeptide having gamma-interferon activities that could be produced conveniently by recombinant DNA or synthetic organic techniques;
These and other objects of the present invention will be apparent to those skilled in the art in light of the present descriptions, accompanying claims, and appended drawings.